Purpose or scope: A role for somatic mutations in carcinogenesis and genetic disease is well accepted, but the degree to which mutation rates influence cancer initiation and development is under continuous debate. Recently accumulated genomic data has revealed that thousands of tumor samples are riddled by hypermutation, broadening support that many cancers acquire a mutator phenotype. This major expansion of cancer mutation datasets has provided unprecedented statistical power for the analysis of mutation spectra, which has confirmed several classical sources of mutation in cancer, highlighted new prominent mutation sources and empowered the search for cancer drivers. In our work we combined mechanistic knowledge obtained through our experiments with yeast models to interrogate the large whole-genome datasets of cancer mutations in order to gain mechanistic insight for understanding the impact of mutations on cancer and genetic disease. Research subject: We combined analysis of mutation signatures of human APOBEC3A and APOBEC3B separately expressed in yeast. The mutation signatures defined in the yeast experiments were then applied to interrogation of large datasets of mutations in human cancers Significant materials equipment or methods: Analysis of genome-wide mutation spectra was empowered by a package of flexible analytical tools that can be configured by researchers to test specific hypotheses about mutagenesis patterns within a complex mix of mutagenic processes operating throughout the history of individual cancers. Specific version of this package is set for identifying cancer samples with APOBEC mutagenesis pattern. This analysis, called Pattern of Mutagenesis by APOBEC Cytidine Deaminases (P-MACD), has been recently integrated into the Broad Institute TCGA GDAC Firehose and is currently available for Firehose standard and customized runs. It allows users to explore correlations of APOBEC mutagenesis with multiple clinical and molecular features, e.g., gene expression and hotspots in significantly mutated. Another critical component in our research was expression of human APOBEC enzymes in a yeast reporter strain (deleted for uracil glycosylase) that generates chromosomal ssDNA upon temperature shift. Telomere uncapping in the presence of ssDNA-damaging mutagens results in selectable mutation clusters inactivating multiple reporter genes. Importantly, resection of the complementary strand precludes excision repair and uracils from cytidine deaminations gave rise to C T transitions, which marks positions of all cytidine deaminations made by an APOBEC enzyme Accomplishments: An antiviral component of the human innate immune system - the APOBEC cytidine deaminases was recently identified as a prominent source of mutations in cancers. We investigated the distribution of the APOBEC-induced mutations across the genomes of breast and lung cancer samples. Contrary to the known elevation of mutation rate in late replicating regions, also characterized by reduced chromatin accessibility and gene density, we observed a marked enrichment of APOBEC mutations in early-replicating regions. This unusual mutagenesis profile is likely to be associated with propensity to form a single-strand DNA substrate for APOBEC mutagenesis and should be accounted in statistical analyses of cancer genome mutation catalogues aimed at understanding mechanisms of carcinogenesis and highlighting genes significantly mutated in cancer.